Aberrant light sensing and motility in the green alga Chlamydomonas priscuii from the ice-covered Antarctic Lake Bonney

ABSTRACT The Antarctic green alga Chlamydomonas priscuii is an obligate psychrophile and an emerging model for photosynthetic adaptation to extreme conditions. Endemic to the ice-covered Lake Bonney, this alga thrives at highly unusual light conditions characterized by very low light irradiance (<15 μmol m−2 s−1), a narrow wavelength spectrum enriched in blue light, and an extreme photoperiod. Genome sequencing of C. priscuii exposed an unusually large genome, with hundreds of highly similar gene duplicates and expanded gene families, some of which could be aiding its survival in extreme conditions. In contrast to the described expansion in the genetic repertoire in C. priscuii, here we suggest that the gene family encoding for photoreceptors is reduced when compared to related green algae. This alga also possesses a very small eyespot and exhibits an aberrant phototactic response, compared to the model Chlamydomonas reinhardtii. We also investigated the genome and behavior of the closely related psychrophilic alga Chlamydomonas sp. ICE-MDV, that is found throughout the photic zone of Lake Bonney and is naturally exposed to higher light levels. Our analyses revealed a photoreceptor gene family and a robust phototactic response similar to those in the model Chlamydomonas reinhardtii. These results suggest that the aberrant phototactic response in C. priscuii is a result of life under extreme shading rather than a common feature of all psychrophilic algae. We discuss the implications of these results on the evolution and survival of shade adapted polar algae.


Introduction
Light provides energy and information that regulates many cellular processes in plants and algae. Motile green algae have sensitive mechanisms for light detection and can induce movement across a light gradient, either toward (positive phototaxis) or away from a light source (negative phototaxis). 1 Phototaxis is regulated by a specialized organelle called an eyespot that allows for the precise detection of light intensity and direction. 1,2 Photoreceptors are critical components of the light-sensing apparatus that controls phototaxis and a plethora of other processes including photosynthesis, circadian rhythms, and gametogenesis. [3][4][5][6] Phototaxis has evolved independently multiple times in diverse microbial lineages including cyanobacteria, algae, and protists, [7][8][9] suggesting that lightdirected movement confers an evolutionary advantage that allows free-swimming microbes to avoid stress from either insufficient or excess light.
Many important insights on light sensing come from model species, such as Chlamydomonas reinhardtii, 10 but green algae are found in diverse habitats. Many environments are populated by species thriving under environmental regimes that are untenable for growth of most model algae. Lake Bonney of the McMurdo Dry Valleys in Antarctica is one such environment. Microalgal communities in this lake are challenged with perpetual low temperatures, extreme shading under a perennial ice cover, prolonged periods of darkness during the polar winter, nutrient deficiencies, supersaturated oxygen levels, and high salinity. 11,12 The ice cover prevents wind-driven mixing and environmental inputs, making this lake an unusually stable and highly stratified environment, often termed a "natural laboratory" for the study of extremophilic biology. 12 Lake Bonney is a home to a diverse algal community, including one of the best studied polar chlorophytes Chlamydomonas priscuii, recently re-named from Chlamydomonas sp. UWO241. 13 Chlorophytes dominate the phytoplankton communities of Lake Bonney. C. priscuii has only been detected in the deep photic zone at 17 meters below the surface of the ice 14 while a second chlorophyte, Chlamydomonas sp. ICE-MDV is found throughout the photic zone and is the dominant chlorophyte within the shallower, under-ice layers. 15 In its natural environment C. priscuii is exposed to year-round low temperatures (~4°C), hypersalinity (700 mM NaCl), low light irradiance (<15 µmol m −2 s −1 ) with a narrow spectral range (450-550 nm), and long periods of darkness during the polar night. 14 C. priscuii is an obligate cold extremophile (psychrophile) that experiences heat shock and cell death at temperature >18°C. 7,16 Under lab conditions, C. priscuii is present as either biflagellate, highly motile single cells or as nonmotile, multi-celled palmelloids. 17 observed a very small eyespot composed of a single layer of carotenoid-rich globules. Positive phototaxis was only possible at higher temperatures (25°C) but not at those closer to its natural environment (7°C). 17 In native phytoplankton communities, 18 observed that while there is no evidence of diel migration in the water column, shallow phytoplankton populations exhibited positive phototaxis, while deeper communities (12 m and 20 m sampling depths) did not. The spatial distribution of different Chlamydomonas populations within the water column of Lake Bonney, combined with recent advances in the study of C. priscuii, including the sequencing of its genome, 19 prompted us to further investigate its phototactic response.

Materials and methods
Strains and Growth Conditions: Chlamydomonas priscuii (previously UWO241, CCMP1619) was originally isolated in early 1990s from the deep photic zone (17 m sampling depth) of the east lobe of Lake Bonney, Antarctica. 14 Chlamydomonas sp. ICE-MDV was originally isolated in 2014 from an enrichment culture of the east lobe of Lake Bonney. 20 Chlamydomonas reinhardtii (CC-1690) was obtained from the Chlamydomonas Resource Center. All cultures were grown axenically in Bold's Basal Medium (BBM) supplemented with 700 mM NaCl at 4°C (C. priscuii), 70 mM NaCl at 4°C (ICE-MDV), or 0.43 mM NaCl at 24°C (C. reinhardtii). All cultures were grown in 500 mL Erlenmeyer flasks continuously aerated with ambient air filtered through a 0.2 µm filter and under continuous light (40 µmol m −2 s −1 ) provided by full spectrum LED light bulbs. Light intensity was measured with a quantum sensor attached to a radiometer (Model LI-189; Li-COR). Cell growth was monitored as change in optical density at 750 nm and cell density was measured using a Countess II FL Automated Cell Counter (ThermoFisher Scientific). To ensure that the cultures were viable at the time of the experiment, cell death was measured by labeling with the fluorescent dye SYTOX Green (ThermoFisher Scientific) as described previously. 16 To exclude cell mortality as the reason behind the lack of phototaxis, we ensured close to 100% cell viability in each experiment (data not shown). Unless otherwise specified, actively growing cultures in the mid-log phase were used in all experiments. Images of algal cells were taken using a Zeiss Axiophot Microscope (Carl Zeiss AG) on a wet mount slide.
Identification of photoreceptors genes in green algal genomes: The C. priscuii genome [19, BUSCO score of 85%] and transcriptome 7 were recently sequenced. These datasets were screened for the presence of photoreceptor genes using previously identified sequences from C. reinhardtii and conserved photoreceptor domains 21 obtained from Phytozome (v6.1) as queries. 22,23 Photoreceptor genes in C. priscuii were identified through a tBLASTn search (e-value<e −10 , bit-score>100) and manually inspected for redundant sequences and to ensure correct gene structure annotation. Conserved domains typical for photoreceptors were identified in the C. priscuii genome using Pfam 24 and NCBI Conserved Domain Database. 25 The genomes of closely related species from the order Chlamydomonadales were obtained from PhycoCosm 26 and included: Chlamydomonas eustigma NIES-2499, 27 Chlamydomonas incerta SAG7.73, Chlamydomonas schloesseri CCAP 11/173, Edaphoclamys debaryana CCAP 11/70, 28 Dunalella salina CCAP19/18, 29 Gonium pectorale NIES-2863, 30 and Volvox carteri v.2.1. 31 The genome of the only other psychrophilic Chlamydomonadalean alga available, Chlamydomonas sp. ICE-L 32 was obtained from GenBank. Photoreceptor genes identified in genome of ICE-L were identical at the nucleotide level as those found in the genome of ICE-MDV, 33 suggesting that these strains belong to the same species. Multiple sequence alignments were performed using ClustalW 34 implemented through Geneious Prime (Biomatters Ltd, Auckland, New Zealand).
Phototaxis dish assay: Phototaxis dish assay was performed according to the protocol by 35,and 36. Cells from exponentially growing cultures (~6x10 6 cells/mL] were resuspended in phototaxis buffer (5 mM HEPES, 0.2 mM EGTA, 1 mM KCl, 0.3 mM CaCl; pH 7.2). In all cases, the phototaxis buffer was supplemented with NaCl (0.43 mM, 70 mM, 700 mM) to match the growth conditions for each species. The algae were incubated for 30 minutes under dim red LED light (624 nm; Cree, Inc.) at the growth temperature to promote motility. Phototaxis was observed in a petri dish (35 mm diameter, 10 mm thickness) placed in a dark chamber and illuminated with a unilateral blue (470 nm; Cree, Inc) or green (525 nm; Broadcam Limited) LED light at 1 or 10 µmol m −2 s −1 for 5 minutes (C. reinhardtii) or 15 minutes (C. priscuii and ICE-MDV). ROS and their quenchers were previously shown to regulate the phototactic sign in C. reinhardtii. 37 We used H 2 O 2 (12.23 mM) to induce positive phototaxis and dimethylurea (DMTU; 75 mM) as quencher of H 2 O 2 to induce negative phototaxis. Unless otherwise specified, all experiments were performed at a temperature corresponding to the growth conditions for each species (24°C for C. reinhardtii; 4°C for C. priscuii and ICE-MDV). The plates were photographed before and after incubation, and the resulting images were used to compare cell movement. All assays were completed at a minimum of three biological replicates.
The direction and strength of the phototactic response was quantified by determining pixel intensity in the images using ImageJ 38 according to a previously described protocol 36 with modifications. In brief, images of the same plate before and after the application of unidirectional light were converted to grayscale and color inverted. The 'before' image was subtracted from the 'after' image. The pixel density of the entire dish (total density) and the half of the dish closest to the light (phototactic cell density) were measured. The phototactic index was calculated as (phototactic cell density)/(total density). A phototactic index of 1 represents a strong positive phototactic response (movement toward the light), a phototactic index of 0 represents a strong negative phototactic response (movement away from the light), and a phototactic index of 0.5 represents no phototactic response (no directional movement in response to light).
Photoshock assay: The photoshock response was observed according to the protocol described in 35,and 36,on a Zeiss ApoTome microscope equipped with a camera (Carl Zeiss AG). Cells were resuspended in phototaxis buffer at a concentration of ~1x10 6 cells/mL and incubated in dim red LED light (624 nm) at their growth temperature as described above. To observe non-directional motility, cells were observed at 60 µmol m −2 s −1 white light. To observe photoshock, cells were observed at dim red light and shocked with a rapid flash of bright white light using a Speedlight 270EX II external flash (Canon Inc.). Videos were created by taking a 15-second series at 62 frames/s and processed by Zen Pro (Carl Zeiss AG). ImageJ was used for video quantification and statistical analysis were performed in RStudio. All assays were completed at a minimum of three biological replicates, with at least 9 fields of view analyzed for each species.

Results and discussion
The psychrophile C. priscuii has a reduced repertoire of photoreceptor genes Screening the nuclear genome of C. priscuii revealed only eight (8) full-length photoreceptor genes, a reduced complement compared to most of its green algal relatives (Figure 1a). These genes contained all conserved domains typical for photoreceptors, suggesting functional proteins (Figure 1b). In contrast, the genome of C. reinhardtii encodes for at least 15 photoreceptor genes, 21 which are conserved within the Chlamydomonadales and most species examined within this group encode 14-16 full-length genes. This includes Chlamydomonas sp. ICE-L, an Antarctic sea ice alga and a strain of ICE-MDV ( Figure 1a; Table S1).
Our results suggest that C. priscuii encodes a single channelrhodopsin gene (ChR1], in contrast to two genes in the C. reinhardtii genome. The ChR photoreceptors are lightgated cation channels, 40 and are key determinants of phototactic responses in C. reinhardtii. 6,21,[41][42][43] The sites for retinal binding, which are involved in light sensing and protein conformational change 44 are conserved in C. priscuii, suggesting a functional photoreceptor ( Figure S1). 42,proposed that ChR hyperphosphorylation is an important component of phototactic signaling in C. reinhardtii but phosphosites are poorly conserved outside of the Reinhardtinia clade with only three predicted phosphosites in the C. priscuii ChR compared to twelve in ChR1 in C. reinhardtii ( Figure S1]. Furthermore, none of the conserved phosphosites were in the C-terminal region of the C. priscuii ChR, which is of key importance for light-induced ChR1 hyperphosphorylation in C. reinhardtii. 42 Whether these features in the sequence of the gene affect the function and regulation of ChR in C. priscuii remains to be experimentally examined. The gene encoding plant-like cryptochrome (pCRY), that entrains the algal circadian clock in C. reinhardtii 3 was not detected in the C. priscuii genome despite being present in all other green algal genomes examined here. The circadian rhythm in C. priscuii has not been examined yet, but in its natural environment this alga has a very unusual photoperiod with months-long periods light and dark. 45 It appears that C. priscuii retains the animal-like cryptochrome (aCRY) and one copy of the phylogenetically conserved Drosophila, Arabidopsis, Synechocystis, Homo (DASH) cryptochrome (CRY-DASH), both of which regulate the transcription of genes involved in photosynthesis, chlorophyll biosynthesis, and maintenance of efficient photoautotrophic growth. 5,46 We could also detect only two histidine-kinase rhodopsin genes (COP5 and COP8) in the genome of C. priscuii. This in contrast to at least six to eight genes in other green algae (Table  S1), including C. reinhardtii (COP5-12). The function of histidine-kinase rhodopsin photoreceptors is not well understood in algae. 47,48 We detected two blue-light receptor phototropins genes (PHOT1 and PHOT2) in the C. priscuii genome. Both PHOT genes have the conserved Light-Oxygen-Voltage (LOV) and Ser/Thr kinase domains (Figure 1b) important for blue-light sensitivity and signal transduction. 49 ICE-L and C. eustigma also share this feature. Having two PHOT receptors is typical for land plants, 50 but most unicellular green algae examined to date, including C. reinhardtii and its close relatives (Figure 1a) encode a single PHOT receptor. The C. priscuii nuclear genome has a high degree of gene duplications (highest of any chlorophyte studied to date), particularly for genes involved in light harvesting and photosynthesis. 19 Gene duplication is increasingly being viewed as a means of adapting to harsh conditions. 51 It was hypothesized that C. priscuii has retained genes important for life in its cold and shaded environment. 19 PHOT is involved in the induction of nonphotochemical quenching at high light intensities and is hypothesized to aid in photoprotection. 4

The Antarctic C. priscuii is motile but exhibits weak photobehaviours
We examined the ability of C. priscuii to move in response to light signals and compared it to that of the well-studied responses in the model C. reinhardtii. We also tested the phototactic behavior of its close relative ICE-MDV, isolated from the shallow photic zone in Lake Bonney. 20 Natural PAR levels experienced by ICE-MDV (~50 μmol m −2 s −1 ) are approximately fivefold higher compared to that of C. priscuii (<15 μmol m −2 s −1 ) 52 allowing for a direct comparison between two closely related psychrophiles from the same environment but adapted to different light conditions. Both psychrophiles exhibited small eyespots, compared to that observed in C. reinhardtii (Figure 2a). All algal species had two flagella ( Figure 2a) and we confirmed motility under non-directional white light (Movie S1-S3); however, we show that the two  Antarctic species swim slower than the mesophilic C. reinhardtii (Figure 2b). Using a dish phototaxis assay, we demonstrated that C. priscuii exhibits a weaker photo behaviour compared to both ICE-MDV and C. reinhardtii. We first exposed algal cultures to unidirectional green light (λ = 525 nm) that regulates phototaxis and has minimal impact on photosynthesis. 35 This wavelength induced prominent and rapid negative phototaxis in C. reinhardtii after 5 min, even at very low light intensity (1 µmol m −2 s −1 ) (Figure 3a, 3b). To account for the slower swimming speeds, we exposed the Antarctic species to this treatment for 15 minutes. We observed strong and consistent positive phototaxis in ICE-MDV after 15-minute exposure to green light even at very low light intensity (1 µmol m −2 s −1 ) but the same treatment induced a much weaker and inconsistent phototaxis or no phototactic response in C. priscuii (Figure 3a, 3b). We also tested for phototaxis under blue light (λ = 470 nm), the predominant wavelength in the depths of Lake Bonney. Once again, we observed weak or no phototaxis in C. priscuii. ICE-MDV exhibited a strong positive phototaxis under blue light, whereas C. reinhardtii also moved rapidly but away from the light ( Figure S2). These results suggest that C. priscuii exhibits a weak and inconsistent phototactic behavior under environmentally relevant light conditions, compared to a robust response in both C. reinhardtii and ICE-MDV.
Previous work on C. reinhardtii has suggested that phototactic behavior consists of three steps: 1) photoreception by ChR; 2) a signal transduction pathway that involves Ca 2+ and reactive oxygen species (ROS); and 3) a change in the beating balance between the two flagella that regulates the phototactic turning. 43 Thus, the weak or absent phototaxis observed in C. priscuii could be a result of a defect in one or more of these steps. To test for flagellar defects, we tested the photoshock response in C. priscuii. This ChR-mediated response occurs when algae sense a sudden and strong illumination, which causes a brief stop (<0.5 s) and/or a period of backward motion. 41 It has been documented that the C. reinhardtii mutants ptx1 and lsp1 are not phototactic due to flagellar defects but display clear photoshock response as a result of a functional light-sensing and signaling apparatus. 53 Thus, a lack of a photoshock response would suggest a decreased ability to sense light signals.
A microscope-based photoshock assay revealed that C. priscuii has a very weak photoshock response where <10% of cells stopped or reversed their swimming direction when exposed to a very brief (2 ms) bright light flash (Figure 4; Movie S4). In contrast, we observed a robust photoshock response in C. reinhardtii (>98% responsive cells) (Figure 4; Movie S5). ICE-MDV displayed a photoshock response, albeit not as strong as the one observed with C. reinhardtii (~65% responsive cells) (Figure 4; Movie S6). These result, and the demonstrated ability of C. priscuii to move under nondirectional light (Movie S1), suggest that this species has functional flagella but aberrant light-sensing ability.

The regulation of phototaxis in psychrophilic algae
The direction of phototaxis is redox regulated in C. reinhardtii, 37 and we tested whether the same is true in its psychrophilic relatives. As shown previously, addition of H 2 O 2 to the culture media caused positive phototaxis in C. reinhardtii exposed to 10 µmol m −2 s −2 green light, while reactive oxygen species (ROS) scavenging by dimethyl thiourea (DMTU) caused negative phototaxis ( Figure 5). The mechanism behind ROS involvement is poorly understood, but it has been postulated that phototactic behavior maintains a moderately reduced state of the cytoplasm and high photosynthetic activity under variable light conditions. 37   Our results suggest that psychrophilic algae employ different mechanisms of phototaxis regulation compared to the model C. reinhardtii. Addition of H 2 O 2 had a minimal effect on both Antarctic species ( Figure 5). The regulation of phototaxis by H 2 O 2 appears to be light quality-and quantitydependent, as all three species displayed weak or absent phototaxis under blue or low intensity green light ( Figure  S3a). ROS scavenging by DMTU resulted in a very weak positive or no phototactic response in C. priscuii but it induced a strong positive phototaxis in ICE-MDV at all light conditions tested here (green and blue light, 1-10 µmolm −2 s −1 ) ( Figure 5, Figure S3b). Overall, our results suggest that the mechanism behind ROS-dependent regulation of phototaxis is species specific. A recent paper reported that C. priscuii has a high capacity for ROS detoxification through constitutive upregulation of the ascorbate pathway. 13 Next, we examined the role of temperature on the strength of the phototactic response in green algae. Previous work by 17,observed strong phototaxis in C. priscuii cultures in the stationary phase at 25° exposed to high intensity white light, suggesting that the phototactic ability of this alga depends on temperature. To test this, we performed a dish phototactic assay with all three species at suboptimal temperatures (4°C for C. reinhardtii; 24°C for C. priscuii and ICE-MDV) at 10 µmol m −2 s −1 of green light, and in the presence of H 2 O 2 and DMTU. All species were incubated at the experimental temperature up to 2 hours prior to the experiment. In C. reinhardtii and ICE-MDV, increased time at suboptimal temperatures lead to a decrease in the phototactic response ( Figure S4). We also did not observe a phototactic response in C. priscuii, even after 2 hours at 24°C. To test whether the culture growth stage has an effect on motility, we also performed a dish phototaxic assay at the stationary phase for all three species, and we show a decreased motility in C. reinhardtii and an absence of motility for ICE-MDV and C. priscuii in stationary phase ( Figure S5) when compared to actively growing cultures in the mid-log phase ( Figure 3). Thus, neither increased temperature nor a different growth stage induced strong phototaxis in C. priscuii under environmentally relevant light conditions. Taken together our results suggest that the Antarctic alga C. priscuii has functional flagella but a weak and inconsistent ability to perceive and translate light signals into phototactic motion, regardless of the intensity or quality of light, presence of ROS, temperature or growth stage. We suggest that this is a result of life under extreme shading rather than a common feature of all psychrophilic algae since its close relative ICE-MDV displays a robust ability to move in response to light. Moreover, the high salinity gradient in the deep photic zone of Lake Bonney restricts natural phytoplankton populations from vertical movement in the water column. 54 This fits with earlier studies on native phytoplankton communities in Lake Bonney that demonstrated that shallow water phytoplankton populations had a strong positive phototactic response while deeper water populations had a weak or no phototactic behavior. 18

Keeping the status quo in Lake Bonney
So, what does this mean for the lifestyle of C. priscuii in the deep photic zone of salty Lake Bonney? Our results support the hypothesis that, under physiologically relevant conditions (low bluegreen light, high salinity, and low temperature), C. priscuii has a limited capability to perceive and translate light signals into rapid motility toward areas of increased light within the water column. This could be due to its small eyespot, reduced photoreceptor repertoire, or altered ROS signaling mechanisms downstream of light perception. A detailed examination of the size and composition of the eyespot, the activity of the psychrophilic ChR photoreceptor, and the signaling pathways that lead to flagellar motility will shed light on phototaxis in shade-adapted green algae.
The extreme conditions in Lake Bonney have undoubtedly shaped the physiology of C. priscuii beyond phototaxis. This alga is unable to grow at light intensities >250 µmol m −2 s −1 , 11 and lacks short-term photoacclimation response for balancing light energy distribution between the photosystems via state transitions, which is well conserved in many other algal species. 55,56 Instead, it accumulates a unique PSIcyt b 6 f photosynthetic supercomplex, 57 which supports constitutively active cyclic electron flow (CEF) around PSI for energy homeostasis and photoprotection. 58 These features have been associated with a rewired primary carbon metabolism leading to constitutively high levels of stress-related compounds (e.g., sucrose, proline, and antioxidants) that support robust growth under extreme conditions. 7,13,58 It is likely that the combined pressures of low temperatures, hypersalinity, and extreme shading drive these adjustments in its physiology, including a reduced phototactic response. Examination of the evolutionary history of C. priscuii and its arrival in Lake Bonney would further inform on its unique physiology and behavior. Lake Bonney is highly stratified and nutrient limited, particularly for phosphorus. 18,59 proposed that nutrient limitation, rather than light, may drive the tactic behavior of phytoflagellates residing in the deep photic zone. They postulated that motile phytoflagellates have a competitive advantage over nonmotile algae for maintaining their position in the photic zone at discrete depths in response to trade-offs between light utilization efficiency and nutrient availability. Indeed, in nutrient bioassay experiments, Lake Bonney Chlamydomonas spp. were highly competitive under phosphorus-supplemented treatments. 60 Recent models suggest that polar environments will be strongly affected by climate change and ice-free conditions may disrupt the lake stratification, nutrient and light availability. 61 For instance, climate-driven high stream flow years carry significant levels of nitrogen and phosphorus to the nutrient-poor waters of Lake Bonney. 62 How these complex climate-driven processes disrupting the 'status quo' of Lake Bonney will affect the endemic and highly specialized psychrophiles, such as C. priscuii and ICE-MDV, is currently not known. Studying the physiology and environmental responses of these unique organisms is important for predicting their survival in the face of global climate change.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This project was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants awarded to MC. The authors are grateful for the support from the Canada Foundation for Innovation (CFI) and University of Ottawa start-up funding. MP was supported by Ontario Graduate Scholarship (OGS) and Polar Knowledge Canada Antarctic Doctoral Scholarship. PO was supported by OGS and NSERC Canada Graduate Scholarships. RMK was supported by the U.S. Department of Energy (DOE), Office of Science, Basic Energy Sciences (BES) under Award # DE-SC0019138.The authors report there are no competing interest to declare.

Data availability
The genomic data that support the findings of this study are available in Phytozome (https://phytozome-next.jgi.doe.gov/) and NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/). All accession numbers for the sequences are available within the Supplementary Data (Supplementary  Table S1). All other data that support the findings of this study are available from the corresponding author upon reasonable request.